The overall goal of this proposal is understand the cellular mechanisms that mediate the anabolic response of bone to mechanical loading. Mechanical loading of bone induces the movement of interstitial fluid within the spaces inside bone. The resulting mechanical stimulation of osteoblasts and osteocytes caused by fluid shear stress (FSS) is hypothesized to play a role in the response of bone to mechanical loading. Support for this hypothesis comes from the observation that osteoblasts and osteocytes that are stimulated by FSS in vitro exhibit increased metabolic activity compared to cells that are maintained under static culture conditions. The central focus of this application is to determine the cellular mechanisms that mediate the mechanical sensitivity of bone cells to their local environment. Specifically, we propose to examine the role of adhesion sites (commonly referred to as focal contacts or focal adhesions) between osteoblasts and osteocytes to the extracellular matrix that are mediated by integrin cell adhesion molecules in regulating the response of bone cells to FSS. The results of this study should provide an improved understanding of the fundamental cellular and molecular mechanisms that mediate the response of bone cells to mechanical loading by testing the hypothesis that focal adhesions function as mechanoreceptors by coordinating the activity of cytoplasmic signaling molecules that interact with integrins. Physiologically relevant responses by bone cells to unidirectional and oscillatory fluid flow include altered proliferative activity and enhanced differentiation toward an anabolic or osteoblastic phenotype, altered gene expression, increased prostaglandin production, activation of mitogen activated protein kinases (MAPK) and focal adhesion kinase (FAK) and sensitization of the insulin-like growth factor receptor (IGF-1R) to stimulation by IGF-1. Three specific aims are proposed. In Aim 1 we will determine the effect of disrupting the structural integrity of focal adhesions on biochemical response of osteoblasts to FSS. In Aim 2 we will determine the role of focal adhesion kinase (FAK) mediated signal transduction pathways activated by FSS in mechanotransduction. In Aim 3 we will determine the role of the transcription factor NMP4/CIZ in mediating signal transduction pathways activated through focal adhesions in response to fluid shear stress. Together these studies should provide novel information on the cellular mechanisms that mediate mechanotransduction in bone.